Today, there is a growing interest in inserting microelectrodes, microcapillaries and micropipet-tip-sensors into single cells. There is also a growing interest in incorporating sub-micron-sized sensing, sampling, and signal-amplifying particles, as well as large biopolymers into single cells and liposomes. Several ultrasensitive detection and sensing methods are based directly or indirectly on the use of colloidal particles. Examples include quantum dot bioconjugate sensors1,2, the family of Probes Encapsulated By Biologically Localized Embedding (PEBBLE) sensors,3 and silver (Ag) and gold (Au) colloids for use in Surface Enhanced Raman Spectroscopy (SERS) measurements4-6. One of the main limitations for practically using these techniques is the difficulty of noninvasive and quantitative introduction of colloidal particles into the cellular interior7. Furthermore, it would be attractive to direct the introduction of particles into specific subcellular compartments such as the cytosol, nucleus, or even organelles of individual cells.
GUVs are cell-sized liposomes composed of a single lipid bilayer with an entrapped aqueous compartment8. Such liposomes are attractive to use as ultra-small reaction containers in which the reaction under study is confined and separated from the external medium. As such they can be used for studies of biochemical reaction dynamics in compartments mimicking a natural intracellular-intraorganellar environment9-12.
For use as reaction containers, it is necessary to load vesicles with reactants, including biopolymers like DNA and colloid particles or organelles (synthetic or naturally derived). Loading of liposomes can, in principle, be performed by adding the particles during the preparation of the vesicles, since they upon formation trap a part of the medium in which they are formed. The trapping efficiency for small liposomes is, however, limited even for low-molecular-weight compounds and, entrapment of larger structures such as colloids, is of very low probability13,14.
Another approach for liposome-loading is to introduce the materials into preformed vesicles by using micromanipulation-based techniques developed for loading of single cells. One such technique that is feasible to use is the microinjection technique15.
By using microneedles made out of pulled glass capillaries with outer tip-diameters in the range of 200-500 nm, it is possible to penetrate the membrane wall of a liposome, or cell, and eject controlled volumes of a desired reagent inside the vesicle16. Injection volumes are typically in the picoliter to attoliter range and controlled by regulation of injection-time and injection-pressure. The pressure is usually generated by utilization of pressurized-air or oil-hydraulic systems.
All microinjection techniques are based on mechanical permeabilization of lipid membranes. When a mechanical point-load is applied, e.g. by a capillary, onto the membrane of a liposome or cell, the membrane is forced to stretch and the isotropic membrane tension, working in the plane of the membrane, is increased. At sufficiently high membrane tension, the structural integrity of the liposome, or cell, is momentarily lost as holes are formed in the membrane, releasing internal fluid in order to counteract the increase in membrane tension. This membrane rupture occurs at the site of the highest mechanical load, which is the loci where the point-load is applied, thus allowing the insertion of a microinjection capillary into the interior of the liposome or cell.
Whereas microinjection works well with certain cell-types and multilamellar liposomes, there are a few drawbacks to the microinjection techniques with unilamellar vesicles and many cell types. Lipid membrane bilayers are, typically, very elastic and the absence of internal supporting structures in unilamellar liposomes make them very difficult to penetrate by mechanical means. The outer diameter of a tip suitable for injection into thin-walled liposomes and smaller cells is about 200 nm, and the inner diameter is typically in the range of only 100 nm16,17. Such tips are very fragile and extremely difficult to view in a light microscope, making positioning difficult. The main drawback of using small inner-diameter injection tips is, however, the requirement of using ultrapure injection liquids in order to prevent clogging, limiting injection species to solutions of low- and medium-molecular-weight compounds. Micro-injection techniques are considered to be relatively invasive due to the large mechanical forces applied, inducing permanent membrane damage and even lysis of cells and liposomes.
An alternative approach to single-liposome or single-cell loading is to use microelectroporation18. This technique is based on the theory of electro-permeabilization. When exposing a liposome, or cell, to an electrical field, a potential drop is generated across the membrane. At sufficiently high field strength, the critical transmembrane potential Vc, of the membrane is exceeded, and small pores will form in the liposomal/cellular membrane due to dielectric membrane breakdown. The transmembrane potential Vm, at different loci on the membrane of a spherical vesicle during exposure to a homogeneous electric field of duration t, can be calculated fromVm=1.5 rs E cos α(1−exp(−t/τ))where E is the electric field strength, rs is the radius of the sphere, α is the angle in relation to the direction of the electric field, and τ is the capacitive-resistive time constant. Pore formation will occur at spherical coordinates exposed to the largest potential shift, which is at the poles facing the electrodes. Typical value for Vc for a cell-sized vesicle is ˜1V, and the corresponding electric field strength needed for exceeding the critical transmembrane potential Vc, is in the range of 1-10 kV/cm.
In microelectroporation, the analyte to be encapsulated is added to the exterior solution of the liposomes, or cells, and an electrical field is then applied locally, using microelectrodes. The amount of analyte that enters the vesicle is dependent on the analyte concentration gradient, membrane potential, duration of the applied field, and diffusion rate of the analyte19. Drawbacks to the electroporation technique are difficulties of quantitative loading, and loading of structures of sizes larger than the effective pore-diameter, which for electropermeabilized erythrocytes is in the range of 1-to-240 nm20,21. To improve quantitive loading, controlled amounts of analytes can be introduced via a small micropipette tip inserted into a hole pre-formed by electroporation (as described, for example, in JP 8322548). This approach, however, presents a number of disadvantages, including the need to apply a fairly strong electric field (˜1V) to form a hole for tip insertion.
By combining electroporation and the application of a mechanical force onto a membrane vesicle, the strength of the applied electrical field needed for membrane permeabilization may be substantially reduced25. This phenomenon is sometimes referred to as electromechanical destabilization. It has been shown that electrical fields established over lipid bilayer membranes imposes an electrocompressive mechanical stress σe, acting on the lipid membrane. This force works normal to the plane of the membrane and leads to a decrease in membrane thickness. If assuming that a lipid membrane behaves as a capacitor, then the electro-compressive force is proportional to the voltage drop V, over the membrane and thus to the strength of the applied electric field
      σ    e    =            1      2        ⁢                  ɛɛ        o            ·              (                  V                      h            e                          )            where ε is the relative dielectric constant and ε0, is the permitivity and he, is the dielectric thickness of the membrane. The differential overall mechanical work dW, done on the lipid membrane is then simply the sum of the electro-compressive stress σe, and the isotropic membrane tension T, controlled by the amount of mechanical strain applied to the membrane
  dW  =            [                        T          _                +                              1            2                    ⁢                                                    ɛɛ                o                            ⁡                              (                                  V                                      h                    e                                                  )                                      ·            h                              ]        ⁢    dA  where h is the overall thickness of the lipid bilayer membrane, and dA is the change in membrane area. Consequently, when a mechanical strain is applied to a membrane vesicle, the trans-membrane potential needed to achieve permeabilization can be significantly reduced. Therefore this approach for membrane permeabilization may be even less invasive than electroporation since lower electric fields can be used, minimizing the risk of unwanted electrochemical reactions at the membrane surface of a cell or a liposome.